A Feasibility Study Evaluating the use of Circulating Tumour DNA in Genetic tests, as a Proxy for Brain Cancer Biopsy Samples

Circulating tumour DNA (ctDNA) in the plasma represents an exciting analyte for the diagnosis and monitoring of disease in cancer patients. In a number of solid cancers types it has been shown to have clinical utility in diagnostic and molecular profiling, tracking of therapeutic response, monitoring of resistance and tumour heterogeneity and detection of post-surgical residual disease. Such clinical utility would also be of huge value in brain cancer, providing clinical information for patients where it is not currently available and/or providing an option for less invasive biopsy-dependent testing. However, its use in glioma brain cancer patients is thought to represent a particular challenge due to the reduced permeability of the blood brain barrier. Aim: This pilot study sought to investigate the practical aspects and clinical utility of using ctDNA in glioma testing in a diagnostic National Health Service laboratory; whether this analyte could be used as a proxy for the diagnostic and therapeutic decision tests performed as standard on FFPE brain biopsy samples. Additionally, the potential clinical utility of standalone metrics of cell free DNA (cfDNA) concentration was explored. Methods: Peripheral blood samples were collected from a cohort of 39 high grade glioma patients comprised of anaplastic oligodendroglioma, anaplastic astrocytoma and glioblastoma tumour sub-types. cfDNA was extracted using a magnetic bead-based protocol and its quality and quantity assessed by chip-based automated electrophoresis. Where the originating cancers harboured the appropriate biomarkers, samples were run though the Laboratory assays for Isocitrate dehydrogenase gene variant analysis, MGMT promoter methylation status and 1p/19q co-deletion testing. Results: The extraction protocol delivered cfDNA of high purity with a mean of 91% ± 5.5%, within the plasma nucleic acid fraction. For all of the tests performed, results reflected the germline DNA profile rather than the new somatic changes of the tumour. The cfDNA analysis did not pick up the tumour biomarkers seen in the paired tumour biopsy sample. However, in all cases the yield of cfDNA was too low to meet the DNA threshold concentration for the established limit of detection for assays. It was thus not possible to fully explore whether very low levels of circulating tumour DNA could be picked up as a component of the cfDNA. In a second part of the study, cfDNA concentrations for the glioblastoma cohort were assessed in the context of their clinical outcomes data. Within the limitations of the testing strategy, the data showed an interesting correlate, where high cfDNA concentration was independently associated with inferior outcome in terms of overall survival. Conclusions: In spite of the considerable advantages of looking for glioma biomarkers within the cfDNA fraction of plasma, this was currently not possible in our routine diagnostic environment. However, high cfDNA concentration in the glioblastoma sub-cohort showed a correlation with inferior outcome in terms of overall survival. Given the simplicity of obtaining this quantifiable metric, there are grounds for further investigations as to its utility; not only with survival outcomes, but also for correlation with the clinical assessment of tumour burden, blood brain barrier integrity and disease pseudoprogression

Evaluation of high-throughput genomic assays for the Fc gamma receptor locus

Cancer immunotherapy has been revolutionised by the use of monoclonal antibodies (mAb) that function through their interaction with Fc gamma receptors (FcγRs). The low-affinity FcγR genes are highly homologous, map to a complex locus at 1p23 and harbour single nucleotide polymorphisms (SNPs) and copy number variation (CNV) that can impact on receptor function and response to therapeutic mAbs. This complexity can hinder accurate characterisation of the locus. We therefore evaluated and optimised a suite of assays for the genomic analysis of the FcγR locus amenable to peripheral blood mononuclear cells and formalin-fixed paraffin-embedded (FFPE) material that can be employed in a high-throughput manner. Assessment of TaqMan genotyping for FCGR2A-131H/R, FCGR3A-158F/V and FCGR2B-232I/T SNPs demonstrated the need for additional methods to discriminate genotypes for the FCGR3A-158F/V and FCGR2B-232I/T SNPs due to sequence homology and CNV in the region. A multiplex ligation-dependent probe amplification assay provided high quality SNP and CNV data in PBMC cases, but there was greater data variability in FFPE material in a manner that was predicted by the BIOMED-2 multiplex PCR protocol. In conclusion, we have evaluated a suite of assays for the genomic analysis of the FcγR locus that are scalable for application in large clinical trials of mAb therapy. These assays will ultimately help establish the importance of FcγR genetics in predicting response to antibody therapeutics

CpG island methylation and promoter usage in the parathyroid hormone- related protein gene of cultured lung cells

Excessive production of a parathyroid hormone-related protein (PTHrP) by turnouts commonly results in the syndrome of humoral hypercalcaemia of malignancy. We have investigated whether epigenetic changes play a role in over-expression of the PTHrP gene, using cultures lung cells as a model system. Study of the methylation status of CpG dinucleotides in the 5' region of the gene showed that in normal cells the CpG island was completely unmethylated. In the lung squamous cell carcinoma cell line, BEN, two-thirds of the CpG island was substantially methylated. RT-PCR analysis showed that this heavy methylation did not prevent expression of any of the three PTHrP gene promoters. This is a surprising finding, since methylation is usually associated with inhibition of gene activity. Methylation of the 5' non- coding region of the PTHrP gene may not play a role in the regulation of adjacent promoters. Alternatively, maintenance of a demethylated state in the 170 bp at the 3' end of the CpG island may be fundamental for the use of PTHrP promoters.</p